Gammaherpesviruses, including the human gHV Epstein Barr virus and Kaposi's sarcoma associated herpesvirus, infect cells of the immune system and establish lifelong, chronic infection that can exist in a benign, latent state. Decreased immune function (from congenital, acquired or iatrogenic immunedeficiencies) is a risk factor for gHV-associated diseases including tumors and chronic inflammatory disease. Important advances in our understanding of these human pathogens can be made from animal models of infection and disease. We hypothesize that these viral regulatory RNAs serve similar functions in these related gHV, much as divergent viral genes in these viruses target common molecular pathways (such as antigen presentation, cell cycle and apoptosis). We will 1) Define the activity of and requirement for the gHV68 miRNAs in vitro, 2) Test the genetic requirement of viral miRNAs to gHV68 infection and pathogenesis in vivo, and 3) Investigate and identify conserved targets of gHV miRNAs. To accomplish this, we will complete the generation and validation of several mutant viruses, lacking viral miRNAs or lacking a viral target sequence, we will make use of an enzymatically marked virus for identification and purification of infected cells, we will determine the integrated protein changes attributable to the viral miRNAs, and most importantly, we will conduct these studies in the context of whole animal infection. These studies will allow us to definitively address the genetic contribution of virally-encoded miRNAs to in vivo pathogenesis, a question uniquely addressed by the gHV68 model of gHV pathogenesis. These studies are highly likely to provide new insights into gHV miRNAs (e.g. virus alterations suitable for vaccines, identification of targeted pathways for therapeutic restoration, or identification of pathways for small RNA regulation that could be useful in other disease states).